Efficacy of Combination Therapy With The JAK Inhibitor Baricitinib In The Treatment of COVID-19

BACKGROUND: Coronavirus disease-19 (COVID-19), resulting from infection with SARS-CoV-2, spans a wide spectrum of illness. In severely ill patients, highly elevated serum levels of certain cytokines and considerable cytolytic T cell infiltrates in the lungs have been observed. These same patients may bear low to negligible viral burdens suggesting that an overactive immune response, often termed cytokine storm, contributes to the severity of COVID-19. We report the safety and efficacy of baricitinib combined with remdesivir and dexamethasone in 45 hospitalized patients with COVID-19 pneumonia at a tertiary academic medical center. METHODS: Retrospective review of 45 patients hospitalized with COVID-19 pneumonia. Patients received 7-day course of baricitinib, 5-day course of remdesivir and 10-day course of dexamethasone. Clinical status and biomarkers were obtained daily. Outcomes assessed include mortality, duration of hospitalization, presence of shock, need for supplemental oxygen, need for non-invasive ventilation, need for mechanical ventilation and development of thrombosis. RESULTS: Obesity and multiple medical comorbidities were associated with hospitalization in the setting of COVID-19. Treated patients demonstrated rapid declines of C-reactive protein (CRP), ferritin and D-dimer with gradual improvement in hemoglobin, platelet counts and clinical status. Only 2 of 45 (4.4%) treated patients required mechanical ventilation after initiating treatment and there were six deaths (13.3%).Only 2 of 45 (4.4%) treated patients required mechanical ventilation after initiating treatment. There were six deaths (13.3%) and these were associated with lower BMI. CONCLUSIONS: These findings support the utility of immunosuppression via JAK inhibition in moderate to severe COVID-19 pneumonia.

response can promote hyperin ammation and multi-organ immune-mediated pathology (2). This is consistent with ndings of elevated levels of several cytokines in severe cases of other coronavirus infections including Severe Acute Respiratory Syndrome coronavirus 1 (SARS-CoV-1) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) (3,4), as well as murine models of the 1918 in uenza (5).
SARS-CoV-2 suppresses the initial type I Interferon (IFN-I) response that is critical for early control of viral infections (2,6). This allows the virus to escape early immune suppression and to replicate more extensively. When the adaptive immune response is later engaged, profound activation of viral-speci c cytolytic T cells occurs in response to the high viral burden (7). The massive expansion of cytolytic T cells causes considerable tissue damage to virally infected cells and potential damage to other uninfected tissues as innocent bystanders (8-10). This is associated with an elevation of several serum cytokines, such as interferon (IFN)-γ, interleukin (IL)-2, IL-6, IL-10, and the α-chain of the IL-2 receptor (CD25) (11,12), a phenomenon termed cytokine release syndrome. A multi-center, retrospective study of 150 patients with severe COVID-19 showed a strong association between elevated ferritin and IL-6 levels and adverse clinical outcomes (13). These observations have collectively suggested that immunosuppressive therapy might mitigate the severity of COVID-19 infections by reducing cytokine release syndrome as well as inhibiting an overactive cytolytic T cell response.
Various anti-cytokine therapies have been considered for the cytokine release syndrome observed in COVID-19. These include tocilizumab, an IL-6 receptor(R) inhibitor (7,14), anakinra, an IL-1R antagonist (15), and inhibitors of the Janus kinase (JAK) pathways (16)(17)(18)(19). Baricitinib, an inhibitor of JAK1/2, was approved for treatment of rheumatoid arthritis in 2018 (20,21). It inhibits the intracellular cytokine signaling pathways known to be active in severe COVID-19, and reduces levels of IL-2, IL-6, IL-10, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF), some of which are important to the cytolytic T cell response. In February 2020 baricitinib was proposed as a potential treatment for COVID-19 based on arti cial intelligence algorithms (22). The authors hypothesized that baricitinib could directly mitigate the in ammatory response triggered by SARS-CoV-2 infection, and was also identi ed as a numb-associated kinase (NAK) inhibitor with high-a nity for AP2-associated protein kinase 1 (AAK1). AAK1 was previously described as a crucial regulator of clathrin-mediated endocytosis of coronaviruses and other viruses (23). This suggested baricitinib could have direct antiviral effects by preventing virus entry into target cells. This additional mechanism could be complementary to the potential bene ts of inhibiting the cytokine storm associated with severe COVID-19.
In three case series of patients with COVID-19, baricitinib use was associated with improved oxygenation and reduction of certain in ammatory markers (16)(17)(18)(19). The placebo-controlled ACTT-2 study found that baricitinib for 14 days plus remdesivir improved recovery times by one day and reduced 28-day mortality from 7.8-5.1% (16). We report our assessment of the safety and e cacy of baricitinib for approximately 7 days following initiation of treatment with combined remdesivir and dexamethasone in 45 inpatients with moderate to severe COVID-19 pneumonia at a tertiary academic medical center.

Methods:
Patients. Between July 10, 2020, and February 8, 2021, COVID-19-positive inpatients were considered for treatment with baricitinib 4 mg daily (if glomerular ltration rate (GFR) > 60 mL/min), 2 mg daily (if GFR 30-59 mL/min), or 1 mg daily (if GFR 15-29 mL/min) for up to 7 days or hospital discharge, whichever was shorter. Inclusion criteria included a positive SARS-CoV-2 polymerase chain reaction (PCR) test, and one or more of the following: a chest X-ray demonstrating in ltrates compatible with COVID-19 infection, oxygen saturation < 94% on room air, or the need for supplemental oxygen and/or mechanical ventilation.
Exclusion criteria included GFR < 15 mL/min or receiving dialysis, active infection with tuberculosis, absolute lymphocyte count < 200 cells/mL, absolute neutrophil count < 500 cells/mL, and suspected druginduced liver injury. Patients meeting inclusion criteria also received remdesivir 200 mg intravenous (IV) for the rst dose followed by 100 mg IV daily for up to 4 days or hospital discharge, whichever came rst. Patients also received dexamethasone 6 mg IV daily for 10 days or until hospital discharge, whichever was shorter. Forty-ve patients met the criteria and were treated with baricitinib.
Outcomes. Duration of hospitalization, presence of shock, and development of thrombosis during hospitalization were recorded. The presence and duration of supplemental oxygen requirement, noninvasive ventilation, and mechanical ventilation were followed. Patient discharge destination and mortality were also recorded.
Statistics. The start date of baricitinib therapy for each patient was arbitrarily set as day 0. All observations were divided into early and late periods. The early treatment period ran from the date of the rst available laboratory result (generally day − 3) through the day of the rst baricitinib dose (Day 0). The late period ran from the day after starting baricitinib until the last available laboratory result. All statistical analyses were performed in Stata 16.1 (StataCorp, LLC, College Station, Texas, USA).

Patient demographics
Of 45 COVID-19 inpatients treated with baricitinib plus dexamethasone and remdesivir, the mean age was 69.5 years (SD +/-16.7) and 53.3% were female. The average patient was obese (BMI 31), and 84.4% of patients had two or more co-existing medical conditions. The most common medical conditions were hypertension (19%), type 2 diabetes mellitus (15%), hyperlipidemia (11%), autoimmune disease (8%), and obstructive airway disease (8%) ( Table 1). Average duration of baricitinib -(days) 6.0 The mean duration of baricitinib treatment was 6 days, and 44% of patients completed a 7-day course of baricitinib (Table 1). Twenty-two patients did not complete the full 7-day course of baricitinib: 9 patients were discharged before completing the treatment course, medication was discontinued in 8 inpatients in the setting of patient stabilization, 6 patients died, and 1 patient left against medical advice (Tables 1). Three patients completed longer courses of baricitinib based on clinical judgement: one patient completed an 11-day course, one patient completed 12 days, and one patient completed two 7-day courses separated by two days for a total of 14 days. 93.3% of patients started baricitinib within 24-hours of receiving dexamethasone and remdesivir.
At the time of initial presentation, patients had symptoms for an average of 6 days and had a mean aggregate NEWS-2 score of 5 (medium clinical risk requiring an urgent response). Seventy-ve percent of patients had bilateral pneumonia on initial chest X-ray. On admission, 60% of patients required supplemental oxygen, 22% required non-invasive ventilation, and 4% required invasive mechanical ventilation ( Table 2). Table 2 Outcomes among of COVID-19 positive inpatients treated with baricitinib / remdesivir / dexamethasone.
Percentages may not total 100 because of rounding.  Laboratory and clinical variables: In response to starting baricitinib, dexamethasone and remdesivir therapy, hemoglobin reversed its downward trend and increased (Fig. 1A). Platelet counts signi cantly increased and there was no signi cant change in white blood cell count (Fig. 1B). Rising levels of CRP, D-dimer, and ferritin also reversed rapidly and signi cantly after starting baricitinib (Fig. 2). There was no signi cant change in creatinine or ALT, whereas AST declined signi cantly following therapy (Supplemental Fig. 1).
There was also a signi cant improvement in ACTT-CS clinical status scores in response to treatment (Fig.  3). NEWS-2 scores also improved with treatment, although this did not achieve statistical signi cance.

Patient outcomes
The average duration of hospitalization was 11 days. Four patients (8.9%) experienced hemodynamic shock. All patients required supplemental oxygen at some point during their admission and 19 required non-invasive ventilation (42.2%) with an average duration of 5 days. Four patients required mechanical ventilation (8.9%) during hospitalization, with an average duration of 3.75 days. However, only two patients required mechanical ventilation following initiation of baricitinib (one patient was transferred from an outside hospital and a second patient was intubated at time of initial presentation prior to starting baricitinib). 68.9% of patients were discharged home with self-care or home health services. 13.3% of patients were discharged to subacute rehabilitation or a skilled nursing facility. There were 6 deaths (13.3%) (  Discussion: The current ndings support the use of immunosuppressive therapy in moderate to severe COVID-19 using a combination of corticosteroids and a JAK1/2 inhibitor. All patients in this study also received remdesivir and dexamethasone in addition to baricitinib, so it is not possible to assess the individual contribution to recovery of each medication. This study spans a 6-month period during which SARS-CoV2 variants emerged in Vermont, USA. Four patients required mechanical ventilation (8.9%) during their hospitalization. Of these, two received ventilator support prior to starting baricitinib therapy, and one of these was extubated shortly after initiating baricitinib. This contrasts with the average higher mechanical ventilation rate of 14.5% from a meta-analysis of 12,437 COVID-19 ICU admissions (24), and was considerably lower than our own experience in the early phases of the pandemic. The patient population in the current study was equally distributed between males and females, in contrast to many other studies. In addition, half were age 70 or older, which makes the favorable outcome all the more remarkable. Finally, there were six deaths (13.3%) among the 45 patients. Two of these, however, were complicated by sub-massive pulmonary emboli present at the time of admission, prior to initiation of baricitinib.
This study sought to assess the e cacy and safety of 7 days of baricitinib treatment, whereas other recent trials in COVID-19 patients treated for 14 days (16-19). The decision for a shorter treatment period was made out of a desire to balance the suppression of in ammation that might result in tissue damage with an avoidance of prolonged immunosuppression that might delay viral clearance or promote secondary infections. Delays in viral clearance have in fact been observed in other immunocompromised patients, resulting in the emergence of viral variants (25). Additionally, given the known risk of JAK inhibitor-induced thrombosis, in the context of the recognized coagulopathy risk in COVID-19, a shorter treatment course may be favorable and su cient for the duration of cytokine release syndrome in these patients.
Half of the patients with moderate to severe COVID-19 had a BMI greater than 30. This is considerably higher than the 23.2% obesity prevalence for the general population in Vermont (https://www.cdc.gov/obesity/stateprograms/fundedstates/pdf/vermont-state-pro le.pdf). Obesity is a known risk factor for severe COVID-19 infection (26). Obesity is also associated with a baseline in ammatory state (27). Adipose tissue supports the development of tissue resident T lymphocytes that upregulate gene expression for several in ammatory cytokines as well as for cytolytic activity, and express high levels of the checkpoint blocker programmed cell death protein-1 (PD-1) (28). A very similar phenotype of T cells is observed in bronchiolar lavage uid of COVID-19 patients (29). Additionally our retrospective review echoes prior work by the Center for Disease Control that older age is associated with increased risk of hospitalization and poorer prognosis with COVID-19 infection.
No adverse effects were noted from use of baricitinib. In particular, there were no secondary infections.
Despite concern for increased thrombotic risk with baricitinib, we did not observe clinical evidence of new clots during the brief course of baricitinib treatment, although two patients demonstrated signi cant clots on admission prior to initiation of baricitinib.
SARS-CoV-2 is known to suppress the initial IFN-I response, likely through the interaction of particular viral proteins with molecules of the IFN-I signaling pathway (30,31). This allows the virus to rapidly replicate during the early stages of infection. The delayed immune response can then become hyperactive and result in considerable cell death of surrounding tissues. This could include tissues that are not known to support SARS-CoV-2 replication, such as liver in ammation observed in some cases of severe COVID-19 (32). The subsequent release of host RNA and DNA from damaged tissues can strongly activate, respectively, the retinoic acid-inducible gene 1 (RIG-I) and cyclic GMP-AMP synthase (cGAS) nucleic acid sensing pathways, leading to an augmented IFN-I response and persistent in ammation even in the absence of virus. This is consistent with studies showing that death of lung epithelium is due in some instances more to the immune response than to viral-mediated lysis (33). Emerging evidence in animal models of SARS and MERS has revealed that the initial IFN-I response has bene cial effects in the early phases of disease, but may become damaging in the latter phases (34).
Severe COVID-19 has close parallels with other seemingly unrelated syndromes that might collectively be classi ed as hyperin ammatory disorders. Chimeric antigen receptor T (CAR-T) cell therapy exposes patients to a large number of T cells that become activated upon contact with targeted tumor cells, often resulting in a highly in ammatory cytokine release syndrome that can include hypercoagulation and even acute respiratory distress syndrome (ARDS) (35)(36)(37). Toxic shock syndrome is a multiorgan in ammatory syndrome (38) in which tampons infected with Staphylococcus release an enterotoxin that acts as a superantigen by binding both the MHC class II molecule and the β-chain of several T cell receptors (39). This activates a signi cant portion of the T cell repertoire, similar to CAR-T therapy, resulting in injury to many organs including skin, liver, and lung, and can also be associated with coagulopathy and ARDS (38). Consistent with the view of hyperactivation of T cells in these disorders, individuals with HIV and low T cell counts have been noted to have less severe COVID-19 (40).
An additional parallel can be made between severe COVID-19 and hemophagocytic lymphohistiocytosis (HLH). HLH is a severe in ammatory syndrome characterized by fever, hepatitis, spleen and lymph node enlargement, and pancytopenia (41,42). It is often observed secondary to certain viral infections as well as autoimmune syndromes such as juvenile in ammatory arthritis (41). An additional laboratory characteristic is elevated ferritin, which we observed in our severe COVID-19 cases. HLH is likely the result of strong T cell activation producing cytokines that activate macrophages to become highly phagocytic (41,42). Consequently, anti-cytokine therapy has also been used to treat HLH, including IL-1 blockade as well as JAK inhibitors.
In other case series of patients with COVID-19, baricitinib treatment was associated with both an improvement in oxygenation and a reduction in select in ammatory markers (16)(17)(18)(19). The largest of these, the ACTT-2 Study Group, randomized 1033 patients to receive remdesivir and either baricitinib for up to 14 days (515) or placebo control (518). Patients receiving baricitinib had a median time to recovery of 7 days compared to 8 days for the control group, and a 30% higher odds of improvement in clinical status at day 15. Patients receiving high-ow oxygen or noninvasive ventilation at enrollment had a time to recovery of 10 days with combination treatment and 18 days with control. The 28-day mortality was 5.1% in the combination group and 7.8% in the control group.
Limitations of the current study include its retrospective nature, the study site was a single center, and the study did not allow for comparison with untreated controls.

Conclusions:
We report a retrospective review of hospitalized COVID-19 patients treated with a short course of baricitinib in combination with dexamethasone and remdesivir. Consistent with previous literature we report that an elevated BMI and multiple medical comorbidities are risk factors associated with hospitalization following COVID-19 infection. Additionally a higher age, ≥ 2 co-existing medical conditions, and higher illness severity at time of presentation was associated with a poorer prognosis. Baricitinib use was associated with increase in hemoglobin and platelet count, decrease in in ammatory markers (D-dimer, Ferritin and CRP), and improvement in clinical status (speci cally the ACTT-CS). The mean duration of baricitinib use was 6 days, which in light of the observed improvements in clinical status and in ammatory markers, may suggest that a shorter course could be bene cial while reducing the risk of more prolonged immunosuppression and thrombosis. No adverse effects were noted from use of baricitinib. In particular, there were no secondary infections or thrombosis following initiation of baricitinib. The current ndings support the use of immunosuppressive therapy in moderate to severe COVID-19 using a combination of steroids and a JAK inhibitor. Ethics committee review and approval waived as project was retrospective and did not include patient identi ers.
Availability of data: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Consent for publication:
Not applicable Competing interests: The authors declare that they have no competing interests. Funding: This work was supported by National Institutes of Health. Grant number GM118228 (RCB).
Author's contributions: BL assisted in statistically analysis and interpretation of patient data. All authors contributed to writing manuscript. All authors read and approved the nal manuscript Baricitinib plus dexamethasone and remdesivir therapy reverses downward trajectory of hemoglobin and increase platelet count. Daily laboratory results were recorded from routine patient care including hemoglobin (g/dL) [A], white blood cell count (K/cm²) [B], lymphocyte count (K/cm²) [C] and platelet count (K/cm²) [D]. The start date of baricitinib, dexamethasone and remdesivir therapy for each patient was arbitrarily set as day 0. All observations were divided into early (up to day -3) and late periods (up to day +9). Linear regressions were perform for each variable and a difference in slope tested between before treatment (day -3 to day 0) vs. after treatment (day +1 to day +9). early (up to day -3) and late periods (up to day +9). Linear regressions were perform for each variable and a difference in slope tested between before treatment (day -3 to day 0) vs. after treatment (day +1 to day +9). (up to day -3) and late periods (up to day +9). Linear regressions were performed for each variable and a difference in slope tested between before treatment (day -3 to day 0) vs. after treatment (day +1 to day +9).

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